Otitis media is an infection of the middle ear that occurs primarily in children. Left untreated, the disease can result in hearing loss and developmental delays. It is estimated that otitis media accounted for 31 million of the 130 million office visits for respiratory diseases in the period from 1977-87. Recent data indicate that suppurative and unspecified otitis media rank first in the list of the 30 most common diagnoses requiring a physician""s office visit for patients up to age 24. Over one billion dollars per year is spent on treatment of this disease and the related loss of income for working parents is estimated to be between $300 and $600 million. Approximately 83% of all children will have had at least one episode of acute otitis media by three years of age. Non-typable strains of Haemophilus influenzae account for 25-30% of all cases of otitis media, 53% of recurrent otitis media, and are the primary pathogens isolated from 62% of cases of chronic otitis media with effusion. Although non-typable Haemophilus influenzae (NTHi) are primary pathogens in otitis media, neither the pathogenic mechanisms nor the host immunological response has been fully defined for this disease.
Fimbriae, which are surface appendages found on non-typable Haemophilus influenzae, are produced by 100% of the bacteria recovered from the middle ears and nasopharyngeal region of children with chronic otitis media. A vaccine comprised of fimbrin, a filamentous protein derived from the fimbriae of non-typable Haemophilus influenzae was previously developed and is useful in studying, preventing, or reducing the severity of otitis media. However, existing methodologies to isolate fimbrin protein from the bacterial outer membrane are tedious and time-consuming. Similarly, purification of fimbrin expressed by the fimbrin gene in other host vector, is also tedious due to the homology between the fimbrin protein and the outer membrane proteins of the host vector.
A strategy with other bacterial species has been to use an alternative immunogen having relatively short linear peptides. However, such alternative immunogens are of limited value due since antibodies to such alternative immunogens frequently fail to recognize the native pathogen.
It would be desirable to have an immunogenic composition to immunize animals against non-typable Haemophilus influenzae which does not depend on tedious purification techniques.
The present invention provides synthetic chimeric fimbrin peptides which induce an immunogenic response in animals to non-typable Haemophilus influenzae and that do not require tedious purification techniques. The synthetic chimeric fimbrin peptides are synthesized using commercially available peptide synthesizers. Preferably the synthetic chimeric fimbrin peptides are from about 18 to about 55, more preferably from about 35 to about 45, most preferably about 40-41 amino acids. The synthetic chimeric fimbrin peptides comprises three peptide units. The first peptide unit preferably contains 12 to 35, more preferably 15 to 30, most preferably 18 to 19 amino acids and is a subunit of the fimbrin protein. Preferably, the fimbrin subunit is comprised of the amino acids of Sequence ID No. 1 or Sequence ID No. 2. The amino acid of Sequence ID No. 1 is:
Arg-Ser-Asp-Tyr-Lys-Phe-Tyr-Glu-Asp-Ala-Asn-Gly-Thr-Arg-Asp-His-Lys-Lys SEQ ID NO: 1
The amino acid of Sequence ID No. 2 is:
Tyr-Gln-Trp-Leu-Thr-Arg-Val-Gly-Lys-Tyr-Arg-Pro-Gln-Asp-Lys-Pro-Asn-Thr SEQ ID NO: 2
The second peptide unit is a t cell epitope and preferably has from about 14 to about 22, more preferably about 15 to 21, most preferably 16 amino acids. The second peptide unit preferably has the following amino acid sequence:
Leu-Ser-Leu-Ile-Lys-Gly-Val-lle-Val-His-Arg-Leu-Glu-Gly-Val-Glu SEQ ID NO: 3
The third peptide unit is a linker peptide unit which joins the first and second peptide unit. The linking sequence preferably has from about 2 to about 15 amino acids, more preferably from about 2 to about 10 amino acids, most preferably from about 5 to about 6 amino acids. The most preferred amino acid sequence for the linker peptide unit is shown in sequence ID NO. 4. The amino acid of Sequence ID NO. 4 is:
Gly-Pro-Ser-Leu-Lys-Leu SEQ ID NO. 4
The synthetic chimeric fimbrin peptides are useful immunogens against NTHi and also useful as laboratory tools for detecting antibodies in sera. The invention also relates to an immunogenic composition containing the synthetic chimeric fimbrin peptides and a pharmacologically acceptable carrier.